| BACKGROUNDS AND OBJECTIVES:There are 24 members in calcium binding protein S100 family. Most of their genes (including S100A6 gene) locate on chromosome 1q21. Chromosomal rearrangement and gene amplification often occur on this site.S100A6 was firstly found from Ehrlich ascites tumor and has been investigated over 20 years. As a active signal molecule, S100A6 participates some signal pathways to regulate physiological activities of cells and to afftec tumor progression. Recent studies showed that S100A6 expression was up-regulated in melanoma, colon carcinoma, pancreas cancers, gastric cancer, non-small cell lung cancer, most osteosarcoma and hepatic conduit epithelium cancer, and that S100A6 upregulation was related with cancer progress. In molecular level, S100A6 interacts with Sgt1 and melusin to regulate heat shock reaction, interacts with P53 to regulate cell proliferation and apoptosis, binds to RAGE (receptor for advanced glycation end products) to activate JNK and caspases pathways which can induce apoptosis, binds to members of annexin family to regulate cell skeleton and cell migration; interacts with CacyBP/SIP to regulateβ-catenin which is a key factor for tumorigenesis and metastasis.Our previous studies revealed that eexogenetic rhS100A6 could inhibit the proliferation and migration of various tumor cell lines and up-regulate cytoplasmicβ-catenin level; but its effects on normal cells are not available. This study aimed to investigate the effects of eexogenetic rhS100A6 on proliferation, migration andβ-catenin level of 5 transformed cell lines and 2 tumor cell lines.S100A6 expression often is high in various tumors, and has been involved in tumorigenses and development. However, the dynamic changes of its expression during tumor development and its precise role need further investigation. This study established hepatic VX2 transplanted tumor model in rabbit and checked the dynamic alteration of S100A6 expression in serum and metastatic tumor tissue, in order to accumulate experiments data to clarify S100A6 contribution in tumor development.METHODS:1. Preparation of recombinant human S100A6 protein (rhS100A6): pGST-Moluc-hS100A6 was transformed into E coli. BL21; isopropy-β-D-thiogalactoside (IPTG) was used to induce expression of rhS100A6; Glutathione Sepharose 4B Beads was used to isolate and purify fusion protein—GST-S100A6. SDS-PAGE, Western-blotting and BCA assay were performed to quality and quantify.2. Cell proliferation and migration ability. Transformed cell lines HEK293, 9HTE, MEF, CHO, C3H10T1/2 and tumor cell lines VX2 carcinoma and human osteosracoma 143B were treated with rhS100A6 at concentration of 3μg/ml,10μg/ml,30μg/ml,100μg/ml,300μg/ml,1000μg/ml and MTT assay was performed to test cell proliferation. Concentration of 30μg/ml was chosen to perform the subsequent experiments due to the data. Time-dependent effects of rhS100A6 on cell proliferation and migration were tested by MTT, cell scratch assay and Transwell chambers.3.β-catenin level in cytoplasm: 5 transformed cell lines were detected with immunocytochemistry (ICC) and Western-blotting to evaluateβ-catenin levels after intervention of 30μg/ml rhS100A6.4. Establishment of rabbit hepatic VX2 transplanted tumor model: VX2 carcinoma tissue(1 mm X1 mm X1mm) from rabbit was transplanted into rabbit liver. The rabbits after transplantation were carefully cultured and observed.5. Sample collections: experimental rabbits were executed to collect serum and tumor tissue from liver, omentum and lung at 7,12,17,19, 21,23,25,27and 29 days after transplantation. Serum was storaged at -80℃and tissue was made into paraffin sections.6. Enzyme linked immunosorbent assay (ELISA) was performed to measure concentration of S100A6 in serum and immunohistochemistry assay was performed to measure expression of S100A6 in tumor tissues.RESULTS:1. rhS100A6 was successfully prepared.Recombinant protein was performed SDS-PAGE and coomassie blue stain. There was one strip at 36KD and its purity was about 92% by Quanity One. Western-blotting showed that there was a S100A6 positive strip.Concentration of rhS100A6 was about 12.3 mg/ml based on the BSA standard curve(BCA). We harvested 6.15mg rhS100A6 from 1L bacteria solution.2. Exogenetic rhS100A6 had no significant effects on proliferation of 5 tranformed cell lines but inhibited proliferation of 2 tumor cell lines in time- and concentration- dependent manner.1) 5 transformed cell lines were treated with rhS100A6 (3μg/ml,10μg/ml,30μg/ml,100μg/ml,300μg/ml,1000μg/ml). Compared with GST groups, no change was found in their OD values (P>0.05). There were no effects on 2 tumor cell lines in 3μg/ml and 10μg/ml rhS100A6 groups; the other concentration of rhS100A6 decreased the OD value by 19.3%,26.4%,37.8%和47.1% (P<0.05) in VX2 and by 17.4%,23.8%,33.7%和35.2% (P<0.05) in 143B, respectively.2) 5 transformed cell lines were treated with rhS100A6(30μg/ml) and no significant change was found in their OD values in 5 days (P>0.05), compared with their GST groups. However, the OD values of VX2 cells decreased by 13.1%,16.3% and 17.9% (P<0.05) and the OD values of 143B cells decreased by 11.7%,14.7% and 16.6% (P<0.05) in 3d,4d and 5d, respectively.3. Exogenetic rhS100A6 has no significant effect on migration of 5 tranformed cell lines but inhibited migration of 2 tumor cell lines.1) 5 transformed cell lines: (1) Cell scratch assay: after treatment with 30μg/ml rhS100A6, the migration ratio of 5 transformed cell lines has no significant difference with that of GST group (P>0.05). (2)Trsanwell assay: the number of cell penetrated the membrane in rhS100A6 group has no significant difference with that of GST group (P>0.05); (3) furthermore, the OD value of cells penetrated the membrane( eluted from membrane by 33% acetic acid) has no significant difference with that of GST group (P>0.05).2) 2 tumor cell lines: (1) Cell scratch assay: after treatment with 30μg/ml rhS100A6, migration ratio of VX2 cell line decreased by 17.2% and migration ratio of 143B cell line decreased by 16.4% (P<0.05). (2)The result was confirmed by Trsanwell assay: the number of VX2 cell penetrated decreased by 10.1% and the number of 143B cell penetrated decreased by 11.2% (P<0.05);(3) furthermore, the OD value of cells penetrated the membrane( eluted from membrane by 33% acetic acid) decreased by 12.3% (P<0.05).4. Exogenetic rhS100A6 up-regulatedβ-catenin level of 5 transformed cell lines.1) Immunocytochemistry: after treatment with rhS100A6, the optical density of positive stain increased by 25.4% in HEK293 group, 15% in 9HTE group, 22.1% in MEF group, 22.7% in CHO group and 28% in C3H10T1/2 group (P<0.05).2) Western-blotting assay: after treatment with rhS100A6,the value of gray level increased by 11.4% in HEK293 group, 21.9% in 9HTE group, 13% in MEF group, 14.8 in CHO group and 10.1% in C3H10T1/2 group (P<0.05).5. Hepatic VX2 transplanted tumor model in rabbit was successfully founded.45 rabbits were transplanted and all VX2 transplanted tumors were successfully grew (success ratio was 100%). The tumor growth was rapid and the survival time was about 30 days from transplantation. No metastasis was found before 15 days; the metastasis in liver and omentum were found at 17th day, the metastasis in lung was observed at 25th day. Pathological examinations comfirmed that the tumor belongs to squamous carcinoma.6. Concentration of S100A6 in serum progressively decreased in VX2 tumor-burden rabbits.Concentration of rabbit serum S100A6 decreased by 12.1% (P=0.237),20.0%(P=0.105),37.6%(P=0.01),79.9%(P=0.01),89.9%(P=0.01),92.9%(P=0.01),98.3%(P=0.01),98.5%(P=0.010 and 99.0%(P=0.01) from 7th day to 29th day, compared with that of normal rabbit serum and presented significant different form 17th day (the time when metastasis was observed).7. Endogenetic expression of S100A6 progressively decreased in all metastatic tumor tissues, including liver, omentum and lung, in VX2 tumor-burden rabbits.Immunohistochemistry assay found that the degree of S100A6 positive stain progressively decreased and the trend was significant by regression analysis (liver metastasis P=0.01, omentum metastasis P=0.01, lung metastasis P=0.04).CONCLUSIONS:1. High purity rhS100A6 protein can be obtained by transforming pGST-moluc-S100A6 into E coli, inducing its expression with IPTG and purifying it via glutathione (GSH)-agarose beads.. 2. Exogenetic rhS100A6 has no significant effects on proliferation and migration of 5 transformed cell lines(HEK293,9HTE,MEF,CHO and C3H10T1/2).3. Exogenetic rhS100A6 can inhibit the cell proliferation and migration of XV2 tumor cell line and human osteosarcoma cell line 143B.4. Exogenetic rhS100A6 can up-regulateβ-catenin level of 5 transformed cell lines(HEK293,9HTE,MEF,CHO and C3H10T1/2).5. S100A6 concentration of serum sharply decreased at the very beginning of metastasis, and then presents a progressive decrease trend.6. Endogenetic S100A6 expression in metastatic tumor tissues progressively decreases.7. Based on that exogenetic rhS100A6 inhibit proliferation and migration of VX2 cell line and that 100A6 progressively decrease in serum and in metastatic tumor tissues, we can speculate: 1) that 100A6 has the inhibitive effects on tumor and its down-regulation may one of factors which promote tumor development; 2) that 100A6 has the inhibitive effects on VX2 tumor ;2)that the down-regulation of 100A6 may one of factors which promote VX2 tumor development and the tumor progression may be delayed by up-regulating S100A6; and 3) that S100A6 concentration in serum may become a biomaker of VX2 tumor metastasis.
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